HIF-1α介導(dǎo)鐵過載對骨髓紅系造血功能損傷的研究
發(fā)布時(shí)間：2019-03-12 20:30
【摘要】：目的研究鐵過載狀態(tài)下缺氧誘導(dǎo)因子(hypoxia inducible factor-1α,HIF-1α)在骨髓照射損傷鐵過載小鼠骨髓細(xì)胞內(nèi)的表達(dá)情況及其對骨髓紅系造血功能的影響及機(jī)制研究。方法1.將20只C57BL/6小鼠隨機(jī)分成4組,除Ctrl組小鼠外,后3組小鼠均接受4Gy 137Csγ射線照射,照射量率為1.0Gy/min,IO組和RAPA組分別給予腹腔注射右旋糖酐鐵25mg/ml(0.2ml/只),Ctrl組和IR組給予腹腔注射生理鹽水(0.2ml/只),注射4周,IR組和RAPA組鐵累積劑量50mg。RAPA組另予灌胃雷帕霉素4mg/kg體重,每周1次,累計(jì)4周,兩個月后頸椎脫臼處死取材;2.流式細(xì)胞儀檢測骨髓單個核細(xì)胞可變鐵池(labile iron pool,LIP)及骨髓細(xì)胞甩片行普魯士藍(lán)染色驗(yàn)證鐵過載模型。3.半固體甲基纖維素的方法檢測造血干祖細(xì)胞紅系造血集落形成能力:CFU-E(clony-forming units-erythroid,紅系造血集落形成單位)、BFU-E(burst clony-forming units-erythroid,爆式紅系造血集落形成);流式檢測骨髓原始紅細(xì)胞:早期紅細(xì)胞(Ter119+CD71+)、晚期紅細(xì)胞(Ter119+CD71-)的數(shù)量和比例;4.實(shí)時(shí)熒光定量PCR檢測骨髓細(xì)胞HIF-1α及HIF-1α信號通路分子(PI3K/AKT/m TOR)m RNA的表達(dá)水平;ELISA檢測血清EPO的表達(dá)水平。結(jié)果IR組、IO組的HIF-1α較Ctrl組表達(dá)升高,分別是Ctrl組的(1.41±0.09)倍、(2.12±0.17)倍,IO組較IR組表達(dá)升高(P均0.05)。IR組、IO組早期紅細(xì)胞比例較Ctrl組降低,差異無統(tǒng)計(jì)學(xué)意義(P0.05);IR組、IO組的晚期紅細(xì)胞比例較Ctrl組降低,IO組較IR組晚期紅細(xì)胞比例降低,差異具有統(tǒng)計(jì)學(xué)意義(P均0.05)。IR組的BFU-E和CFU-E數(shù)目較Ctrl組明顯減低,IO組的BFU-E和CFU-E數(shù)目較IR組明顯降低(P均0.05)。IR組和IO組HIF-1α相關(guān)信號通路分子(PI3K/AKT/m TOR)較Ctrl組表達(dá)升高(P0.05)。應(yīng)用m TOR抑制劑RAPA使HIF-1α表達(dá)明顯降低,其相關(guān)信號分子表達(dá)也明顯降低,差異有統(tǒng)計(jì)學(xué)意義(P0.05);BFU-E明顯升高,差異有統(tǒng)計(jì)學(xué)意義(P0.05),CFU-E數(shù)目升高,但無統(tǒng)計(jì)學(xué)差異(P0.05);早期紅細(xì)胞和晚期紅細(xì)胞的比例略有升高,無統(tǒng)計(jì)學(xué)差異(P0.05)。結(jié)論1.鐵過載可損傷骨髓的紅系造血功能。通過構(gòu)建鐵過載動物模型發(fā)現(xiàn),鐵過載可以影響鐵過載小鼠的原始細(xì)胞紅系造血集落(CFU-E、BFU-E)形成能力、影響早期原始紅細(xì)胞和晚期原始紅細(xì)胞的比例,尤以對晚期紅細(xì)胞的影響為顯著,照射更加重了這種損傷。2.鐵過載小鼠骨髓單個核細(xì)胞HIF-1α表達(dá)水平明顯升高,提示HIF-1α與鐵過載致骨髓紅系造血功能損傷有關(guān)。研究HIF-1α及其相關(guān)信號轉(zhuǎn)導(dǎo)通路發(fā)現(xiàn),鐵過載使HIF-1α相關(guān)通路信號轉(zhuǎn)導(dǎo)分子PI3K、AKT、m TOR表達(dá)均明顯升高。3.鐵過載對骨髓紅系造血功能的損傷是可逆的。通過應(yīng)用m TOR抑制劑雷帕霉素后,抑制細(xì)胞內(nèi)HIF-1α的表達(dá),可部分恢復(fù)鐵過載對骨髓紅系造血功能的損傷作用。這提示了鐵過載對骨髓紅系造血功能的損傷有望通過抑制HIF-1α來緩解。
[Abstract]:Aim to study the expression of hypoxia inducible factor (hypoxia inducible factor-1 偽 (HIF-1 偽) in bone marrow cells of irradiated mice with iron overload and its effect on erythroid hematopoiesis and its mechanism. Method 1. Twenty C57BL/6 mice were randomly divided into 4 groups. All the mice in the latter 3 groups were exposed to 4Gy 137Cs 緯-ray with the dose rate of 1.0 Gy / min. The mice in IO group and RAPA group were given intraperitoneal injection of iron dextran 25mg/ml (0.2ml/ only), with the exception of the latter three groups receiving 4Gy 137Cs 緯-ray irradiation at a dose of 1.0 Gy 路min ~ (- 1). The rats in Ctrl group and IR group were given intraperitoneal injection of saline (0.2ml/) for 4 weeks. The cumulative dose of iron in IR group and RAPA group was added to 50mg.RAPA group, and the body weight of rapamycin was given once a week for 4 weeks. Two months later, the cervical vertebrae were dislocated and the samples were taken. 2. Flow cytometry for detection of variable iron pool (labile iron pool,LIP in bone marrow mononuclear cells and Prussian blue staining for bone marrow cells flick to verify iron overload model. 3. Hemopoietic colony forming ability of hemopoietic stem progenitor cells was detected by semi-solid methylcellulose method: CFU-E (clony-forming units-erythroid, erythroid colony forming unit), BFU-E (burst clony-forming units-erythroid, burst erythroid hematopoietic colony formation); Flow cytometry was used to detect the number and proportion of early erythrocytes (Ter119 CD71) and late erythrocytes (Ter119 CD71-) in bone marrow. Real-time fluorescence quantitative PCR was used to detect the expression of HIF-1 偽 and HIF-1 偽 signal pathway molecules (PI3K/AKT/m TOR) m RNA) in bone marrow cells and ELISA to detect the expression level of serum EPO. Results the expression of HIF-1 偽 in IR group and IO group was (1.41 鹵0.09) times and (2.12 鹵0.17) times higher than that in Ctrl group, and the expression of HIF-1 偽 in IO group was higher than that in IR group. The percentage of early erythrocytes in IO group was lower than that in Ctrl group, and there was no significant difference (P0.05). In IR group, the percentage of late erythrocytes in IO group was lower than that in Ctrl group, and that in IO group was lower than that in IR group. There was significant difference in the number of BFU-E and CFU-E between). IR group and Ctrl group. The number of BFU-E and CFU-E in IO group was significantly lower than that in IR group (P0.05). The expression of HIF-1 偽-related signal pathway molecule (PI3K/AKT/m TOR) in both). IR group and IO group was significantly higher than that in Ctrl group (P0.05). Application of m-TOR inhibitor RAPA significantly decreased the expression of HIF-1 偽 and the expression of related signal molecules, the difference was statistically significant (P0.05). BFU-E significantly increased, the difference was statistically significant (P0.05), the number of CFU-E increased, but there was no statistical difference (P0.05); early red blood cells and late erythrocytes increased slightly, there was no statistical difference (P0.05). Conclusion 1. Iron overload can damage erythrocytic hematopoiesis of bone marrow. By constructing iron overload animal model, it was found that iron overload could affect the ability of erythropoiesis colony formation (CFU-E,BFU-E) of primordial cells and the ratio of early and late primordial erythrocytes in iron overload mice. In particular, the effect on late red blood cells was significant, and the damage was aggravated by irradiation. 2. The expression of HIF-1 偽 in bone marrow mononuclear cells of iron overloaded mice was significantly increased, suggesting that HIF-1 偽 was related to the damage of erythroid hematopoiesis of bone marrow induced by iron overload. HIF-1 偽 and its related signal transduction pathways were studied. It was found that iron overload significantly increased the PI3K,AKT,m TOR expression of signal transduction molecules in HIF-1 偽 related pathways. 3. The damage of hemopoietic function of erythroid bone marrow induced by iron overload is reversible. By inhibiting the expression of HIF-1 偽 after the application of m-TOR inhibitor rapamycin, the damage effect of iron overload on erythroid hematopoiesis of bone marrow was partially restored. This suggests that the damage of erythrocytic hematopoiesis induced by iron overload may be alleviated by inhibition of HIF-1 偽.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R55
本文編號：2439122
[Abstract]:Aim to study the expression of hypoxia inducible factor (hypoxia inducible factor-1 偽 (HIF-1 偽) in bone marrow cells of irradiated mice with iron overload and its effect on erythroid hematopoiesis and its mechanism. Method 1. Twenty C57BL/6 mice were randomly divided into 4 groups. All the mice in the latter 3 groups were exposed to 4Gy 137Cs 緯-ray with the dose rate of 1.0 Gy / min. The mice in IO group and RAPA group were given intraperitoneal injection of iron dextran 25mg/ml (0.2ml/ only), with the exception of the latter three groups receiving 4Gy 137Cs 緯-ray irradiation at a dose of 1.0 Gy 路min ~ (- 1). The rats in Ctrl group and IR group were given intraperitoneal injection of saline (0.2ml/) for 4 weeks. The cumulative dose of iron in IR group and RAPA group was added to 50mg.RAPA group, and the body weight of rapamycin was given once a week for 4 weeks. Two months later, the cervical vertebrae were dislocated and the samples were taken. 2. Flow cytometry for detection of variable iron pool (labile iron pool,LIP in bone marrow mononuclear cells and Prussian blue staining for bone marrow cells flick to verify iron overload model. 3. Hemopoietic colony forming ability of hemopoietic stem progenitor cells was detected by semi-solid methylcellulose method: CFU-E (clony-forming units-erythroid, erythroid colony forming unit), BFU-E (burst clony-forming units-erythroid, burst erythroid hematopoietic colony formation); Flow cytometry was used to detect the number and proportion of early erythrocytes (Ter119 CD71) and late erythrocytes (Ter119 CD71-) in bone marrow. Real-time fluorescence quantitative PCR was used to detect the expression of HIF-1 偽 and HIF-1 偽 signal pathway molecules (PI3K/AKT/m TOR) m RNA) in bone marrow cells and ELISA to detect the expression level of serum EPO. Results the expression of HIF-1 偽 in IR group and IO group was (1.41 鹵0.09) times and (2.12 鹵0.17) times higher than that in Ctrl group, and the expression of HIF-1 偽 in IO group was higher than that in IR group. The percentage of early erythrocytes in IO group was lower than that in Ctrl group, and there was no significant difference (P0.05). In IR group, the percentage of late erythrocytes in IO group was lower than that in Ctrl group, and that in IO group was lower than that in IR group. There was significant difference in the number of BFU-E and CFU-E between). IR group and Ctrl group. The number of BFU-E and CFU-E in IO group was significantly lower than that in IR group (P0.05). The expression of HIF-1 偽-related signal pathway molecule (PI3K/AKT/m TOR) in both). IR group and IO group was significantly higher than that in Ctrl group (P0.05). Application of m-TOR inhibitor RAPA significantly decreased the expression of HIF-1 偽 and the expression of related signal molecules, the difference was statistically significant (P0.05). BFU-E significantly increased, the difference was statistically significant (P0.05), the number of CFU-E increased, but there was no statistical difference (P0.05); early red blood cells and late erythrocytes increased slightly, there was no statistical difference (P0.05). Conclusion 1. Iron overload can damage erythrocytic hematopoiesis of bone marrow. By constructing iron overload animal model, it was found that iron overload could affect the ability of erythropoiesis colony formation (CFU-E,BFU-E) of primordial cells and the ratio of early and late primordial erythrocytes in iron overload mice. In particular, the effect on late red blood cells was significant, and the damage was aggravated by irradiation. 2. The expression of HIF-1 偽 in bone marrow mononuclear cells of iron overloaded mice was significantly increased, suggesting that HIF-1 偽 was related to the damage of erythroid hematopoiesis of bone marrow induced by iron overload. HIF-1 偽 and its related signal transduction pathways were studied. It was found that iron overload significantly increased the PI3K,AKT,m TOR expression of signal transduction molecules in HIF-1 偽 related pathways. 3. The damage of hemopoietic function of erythroid bone marrow induced by iron overload is reversible. By inhibiting the expression of HIF-1 偽 after the application of m-TOR inhibitor rapamycin, the damage effect of iron overload on erythroid hematopoiesis of bone marrow was partially restored. This suggests that the damage of erythrocytic hematopoiesis induced by iron overload may be alleviated by inhibition of HIF-1 偽.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R55
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